Gene associated with Leishmania parasite virulence

ABSTRACT

The invention relates to the field of combating leishmaniases. Said invention results from the isolation, from wild isolates of  Leishmania major , of a protein-coding gene known as LmPDI which has two regions that are identical to the sequence (Cys-Gly-His-Cys) of the potential active site of the protein disulphide isomerases (PDI). The LmPDI protein is predominantly expressed in the most virulent isolates of the parasite. Said protein forms a novel therapeutic target for developing anti-leishmaniasis medicaments and a novel element that can be used in the composition of immunogenic, and possibly vaccinating, preparations which are intended to protect a human or animal host against  Leishmania.

The invention relates to the field of the fight against leishmaniasis.It results from the identification, from wild isolates of Leishmaniamajor, of a gene coding for a protein, designated LmPDI, having tworegions identical to the sequence (Cys-Gly-His-Cys) of the potentialactive site of protein disulfide-isomerase (PDI). This LmPDI protein ispredominantly expressed in the most virulent isolates of the parasite.It firstly constitutes a novel therapeutic target for developinganti-leishmaniasis drugs, and secondly, a novel element that can formpart of the composition of immunogenic and possibly vaccinatingpreparations intended to protect a human or animal host againstleishmaniasis.

Leishmaniases constitute a heterogeneic group of diseases that affectseveral million individuals and are due to infection of the host by aprotozoic parasite of the genus Leishmania. Clinical expression of theinfection is characterized by a high degree of polymorphism, includingasymptomatic infection, simple or recurring cutaneous forms, diffuse oranergic cutaneous forms, mucocutaneous forms and visceral forms, whichare fatal in the absence of specific treatment. In general, anddepending on the geographical distribution of the disease, each speciesor sub-species of leishmaniasis is responsible for a particular clinicalform; however, this is not a strict rule. Further, in the samegeographical region, the same parasitic species can be responsible forclinical forms of varying severity. This diversity in clinicalexpression of the infection is at least partially due to a diversity inthe virulence of the parasite.

During its cycle, the parasite alternates between two stages: theflagellate promoastigote stage, which is found in the digestive tract ofthe insect vector, and the amastigote stage in the host macrophage.Anti-leishmaniasis drugs are difficult to use, not least because theyare toxic and because of the ever more frequent resistance developed bythe parasite (Lira, Sundar et al, 1999). Further, recently developed andtested vaccines have so far not shown the expected efficacy (Sharifi,FeKri et al, 1998; Khalil, El Hassan et al, 2000).

The absence of tools for controlling leishmaniasis is partiallyexplained by the complexity of the parasite transmission cycles and bythe dearth of current knowledge regarding the biology of the parasite.During the last ten years, several molecules playing a fundamental rolein the biology and infectivity of the parasite have been identified.Modifications to surface glyco-conjugates, particularlylipophospho-glycane (LPG), are associated with modifications to theinfectivity and virulence of the parasite Leishmania (L) major and L.donovani (Beverley and Turco, 1998); Desjardins and Descoteaux, 1998;Sacks, Modi et al, 2000; Spath, Epstein et al, 2000), which does notappear to be the case for L. mexicana (IIg 2000; IIg, Demar et al,2001). Molecules involved in the biosynthesis of LPG: phosphomannoseisomerase (Garami and IIg, 2001), LPG1 (Sacks, Modi et al, 2000; Spath,Epstein et al, 2000), LPG2 (Descoteaux, Luo et al, 1995) and galactosyltransferase (De and Roy, 1999) have also been associated with thevirulence of Leishmania. Other factors in virulence have recently beendescribed. They include the family of cysteine proteases (Mottram,Brooks et al, 1998), mitogen activated protein (MAP)-kinases (Wiese,1998), the A2 gene (Zhang and Matlashewski, 1997), the surfaceglycoprotein gp63 (Chakrabarty, Mukherjee et al, 1996), kinetoplastidmembrane protein (KMP)-11 (Mukhopadhyay, Sen et al, 1998), superoxidedismutase (Paramchuk, Ismail et al, 1997), trypanothione reductase(Dumas, Ouellette et al, 1997), and certain members of the heat shockprotein (HSP) family (Hubel, Krobitsch et al, 1997).

Characterizing virulence factors may have fundamental implications inthe development of novel drugs or vaccines against these diseases.Preferential screening of a protein involved in the virulence of aparasite can avoid the unnecessary appearance of resistance in lessdangerous strains, which resistances may then be transmitted to otherstrains. Further, a mutation of the targeted virulence protein causingresistance to the drug can in that case also cause a reduction or evenloss of the virulence of the parasite and thus have a certaintherapeutic effect.

Over the past decades, a number of approaches have been used to studythe virulence factors for the Leishmania parasite. These approaches werebased on genetic studies, such as the complementation of mutatedparasites (Ryan, Garraway et al, 1993; Descoteaux, Luo et al, 1995;Desjardins and Descoteaux, 1997; Wiese, 1998), the use of the geneinvalidation technique (Titus, Gueiros-Filho et al, 1995; Mottram, Souzaet al, 1996; Dumas; Ouellette et al, 1997; Hubel, Krobitsch et al, 1997;Mottram, Brooks et al, 1998), or the analysis of genes for resistance todrugs on parasites manipulated in the laboratory (Cotrim, Garrity et al,1999; Perez-Victoria, Perez-Victoria et al, 2001). Those studiesidentified several genes that were important to the biology of theparasite, and are currently being validated as targets for novel drugs(Selzer, Chen et al, 1997; McKerrow, Engel et al, 1999), or for thedevelopment and use of attenuated mutants as live vaccines (Titus,Gueiros-Filho et al, 1995; Streit, Recker et al, 2001). It is importantto point out almost all of the studies carried out up to now on thevirulence of the parasite Leishmania are based either on laboratoryclones which have lost their virulence after prolonged culture, or onparasites genetically manipulated by mutagenesis experiments, geneinvalidation or gene overexpression. Thus, it is possible that theconclusions relating to the virulence of the genes identified underthese conditions are not actually relevant to the natural pathogenicityof the parasite in the transmission regions.

With the aim of studying the molecular bases for the virulence of theparasite, avoiding the methodological bias linked to the use oflaboratory strains, the inventors initially isolated wild strains ofLeishmania (L) major with different levels of virulence. L. major is theagent in zoonotic cutaneous leishmaniasis (ZCL), which exists inepidemic proportions in man over a very wide area which extendsseamlessly from Mauritania to Mongolia. The inventors identifiedisolates of L. major obtained from human ZCL lesions, all obtainedduring the same transmission season, and which differ in theirpathogenicity in an experimental model of the infection in BALB/csensitive mice (Example 1). The differences in experimental pathogenicpower correlate with the differences in growth in vitro, which reflectsthe variations in the biology of these wild isolates.

The “differential display” technique (Liang and Pardee, 1992; Liang,Bauer et al, 1995) was then used to identify genes differentiallyexpressed between completely different isolates through theirexperimental pathogenic power in the BALB/c mouse (two highly virulentisolates and two other less virulent isolates). This technique allowsgenes which are expressed at different levels to be studied withoutknowing their sequence in advance. Three transcripts that arepreferentially expressed in the two most virulent isolates were thenidentified. One of these transcripts was completely characterized bydint of screening a cDNA library of L. major. An analysis of thesequence demonstrated a homology with the protein disulfide-isomerasefamily (PDI, Erp60 and Erp72) in eukaryotes. This novel protein has beendesignated LmPDI for the following reasons: like other members of thePDI family, (i) LmPDI possesses two CGHC active regions, (ii) theN-terminal region of this protein contains in a potential signalsequence and, in the carboxy-terminal region, a potential signal forretention in the endoplasmic reticulum (EEDL); (iii) it can organizeitself into an oligomeric structure; (iv) the recombinant proteinproduced in E. coli expresses PDI activity in vitro. Further, outsidethe conserved regions mentioned above, there are very few similiaritiesbetween LmPDI and the other PDIs described above. In fact, the PDIfamily includes a plurality of highly divergent molecules involved inthe maturation of proteins secreted into the endoplasmic reticulum(Noiva 1999; Frand, Cuozzo et al 2000). PDIs are multi-functionalproteins which are involved in complex mechanisms of retention, repair,regulation of expression; they assist changes in conformation to allowonly correctly folded proteins to leave the endoplasmic reticulum. Inaddition to their enzymatic functions (reduction and isomerisation),other functions have recently been attributed to PDIs; they includechaperone activities, the binding of peptides and cellular adhesion(Ferrari and Soling, 1999). It is important to emphasize that LmPDI ispredominantly expressed in the most virulent isolates (Example 2). Intotal, these results suggest that LmPDI plays an important role in thenatural virulence of the Leishmania parasite, and can thus constitute anovel target for chemotherapy or vaccination.

Further, recent data regarding the involvement of the bacterialequivalent of PDIs (Martin, 1995; Ostermeier, De Sutter et al, 1996)(termed DsbA, disulphide bond) make suggestions regarding the role thisprotein could play in the pathogenicity of different micro-organisms (Yuand Kroll, 1999). Deactivation of the DsbA gene dramatically affects thesurvival and virulence of Shigella flexneri (Yu, 1998; Yu, Edwards-Joneset al, 2000). DsbA is also involved in the genesis of the enterotoxin ofVibrio cholerae (Peek and Taylor, 1992; Yu, Webb et al, 1992). DsbA alsoimportant for the pathogenicity of pathogenic Escherichia coli species:in species that are pathogenic for the urinary tract, it catalyses theformation of disulfide bridges of a specific chaperone protein of pilin(Zhang and Donnenberg, 1996). In enteropathogenic species, it is alsorequired for the stability and formation of pili (Hultgren, Abraham etal, 1993; Wang, Bjes et al, 2000).

The present application constitutes the first description suggesting animportant role for PDIs as a virulence factor in a protozoic parasite.LmPDI could exert its effects by assisting changes in the conformationand stability of other factors essentially to the biology andpathogenicity of the parasite Leishmania. Identifying such factors wouldbe extremely advantageous for a better comprehension of the biology ofthis parasite, and for the development of novel treatments or vaccinesagainst leishmaniases.

Thus, in a first aspect the invention concerns a protein involved in thevirulence of Leishmania, comprising at least one (Cys-Gly-His-Cys) siteidentical to the potential active site of a protein from the proteindisulfide-isomerase family (PDI). This protein is preferably a proteincoded by the parasite itself.

In particular, the invention concerns the LmPDI protein of Leishmaniamajor, with sequence SEQ ID No: 3, and any functional variant of LmPDIhaving at least 40% identity, preferably at least 80% identity withLmPDI.

We shall define here a “functional variant of LmPDI” as a protein thatis capable of complementing LmPDI in an infectivity test carried outwith a strain of L. major in which the LmPDI gene has been deactivated.The term “infectivity test” as used here means any test that canevaluate the biological properties relating to the growth of theparasite conventionally associated with virulence. In particular, thethree following types of tests can be cited:

-   -   growth kinetics in a liquid medium of the promastigote form of        the parasite, for example by a technique of the type described        in point 2 of Example 5;    -   the infection capacity of mouse macrophages cultivated in vitro,        using a technique such as that described in Example 6 and in the        article by Kebaïer et al, 2001;    -   the capacity to induce experimental murine leishmaniasis by        infecting sensitive mice (BALB/c, for example). The technique is        detailed in point 3 of Example 5, for example, and in the        article by Kebaïer et al, 2001.

The percentage of identity with LmPDI are evaluated using CLUSTAL Wversion 1.8 software (Thompson J D, Higgins D G and Gibson T J) orBOXSHADE version 3.21 software (Hoffman K and Baron M) which producedpercentage identities of LmPDI with proteins from the PDI family ofseveral species as between 27% and 36% (Example 2).

In a second aspect, the invention concerns a recombinant polypeptidecomprising at least one fragment of more than 10 amino acids of aprotein as defined above, if appropriate fused with a furtherpolypeptide fragment, said recombinant polypeptide being capable oftriggering an immunological reaction against an epitope of LmPDI whenadministered to an animal. The invention also concerns a recombinantpolypeptide comprising at least one fragment of more than 10 amino acidsof a protein as defined above, if appropriate fused with a furtherpolypeptide fragment, said recombinant polypeptide being capable ofbeing recognized by antibodies directed against the LmPDI protein.

Throughout this text, the term “polypeptide” should be taken in itsbroad sense, i.e., including sequences of at least 10 amino acids (ormore when stated), which may or may not comprise glycosylated motifs orglycolipids, and regardless of its primary, secondary or tertiarystructure. The LmPDI fragment present in the recombinant polypeptides ofthe invention described above may be over 15, 20, 30, 50 or 100 aminoacids in size, or even more.

The LmPDI, recombinant or purified from infected cells, and apolypeptide of the invention can be used to immunize a human or animalhost, to protect it from leishmaniasis or to produce and recoverantibodies directed against LmPDI, as described in Example 2.

A particular recombinant polypeptide of the invention is theLmPDI-(His)₆ protein with sequence SEQ ID No: 4, described in Example 2.

A further example of the recombinant polypeptide of the invention is afusion protein between a LmPDI fragment comprising at least one epitopeof LmPDI and a carrier polypeptide contributing to the presentation ofthat fragment to the immune system. It can in particular be a fusion ofall or a portion of the LmPDI with a fragment of β-lactamase, or atetanus or diphtheria anatoxin, or any other polypeptide from apathogenic organism, in particular of parasitic, bacterial or viralorigin.

In a further aspect, the invention concerns a nucleic acid sequencecoding for a protein or a polypeptide as described above. A preferrednucleic acid sequence comprises the sequence coding for LmPDI withsequence SEQ ID No: 2, or a fragment of said sequence with a size of 30nucleotides or more, preferably more than 100 nucleotides, coding for apolypeptide comprising at least one characteristic epitope of LmPDI.

The invention also concerns a nucleic acid vector comprising a nucleicacid sequence of the invention. As an example, it may be a plasmid, acosmid, a phage or a virus. Preferably, a vector of the invention willallow expression in a host cell of a protein or a polypeptide inaccordance with the invention. In particular, a vector of the inventioncan allow expression of LmPDI in a bacterial or eukaryotic cell.

The invention also pertains to a cultured cell comprising a vector asdefined above. Said cell can be a bacterium, a yeast, an insect cell, amammalian cell or any other type of cell. It can be used either toexpress and possibly produce a protein or a polypeptide in accordancewith the invention, or to produce a vector which will then serve toexpress a protein or a polypeptide in accordance with the invention in afurther cultured cell type, or in vivo. Purely by way of non-limitingillustration, CHO, VERO, BHK21 cells and insect cells can be cited ascell types that can be used in vitro in the context of the presentinvention. Similarly, BCG and Salmonella typhimurium can be cited ascells that can be used in vivo. Finally, it is important to note thatadministration to an individual of a viral vector, for example a vaccinevirus or DNA coding for a polypeptide or a protein as described abovefor vaccine purposes is also encompassed within the scope of theinvention.

A particular cell of the invention is the bacterial strain LmPDI-XL₁deposited at the Collection National de Culture des Microorganismes[CNCM, the National Collection of Microorganism Cultures], on 31 Jan.2002 with accession number I-2621. This strain is derived from aXL1-blue MRF′ strain bacterium with genotype Δ(mrcA)183Δ(mcrCB-hsdSMR-mrr)173endA1 sup E44 thi-1 recA1gyrA96 reLA1 lac[F′ proABlac Z ΔM15 Tn10 (Tet′)], transformed by the plasmid pBK-CMV-LmPDI. Thisplasmid corresponds to the plasmid pBK-CMV sold by Stratagene (La Jolla,Calif.) to which cDNA from LmPDI has been added between the EcoRI andXho I restriction sites.

The invention also pertains to a nucleic acid probe which specificallyhybridizes under stringent conditions with the nucleic acid sequence ofSEQ ID No: 2, allowing the presence or absence of the virulence genecoding for LmPDI to be determined in a biological sample.

“Stringent hybridization conditions” are defined herein as conditionsthat allow specific hybridization of two DNA molecules at about 65° C.,for example in a solution of 6×SSC, 0.5% SDS, 5× Denhardt's solution and100 μg/ml of denatured non specific DNA or any solution with anequivalent ionic strength, and after a washing step carried out at 65°C., for example in a solution of at most 0.2×SSC and 0.1% SDS or anysolution with an equivalent ionic strength. However, the stringency ofthe conditions can be adapted by the skilled person as a function of thesize of the sequence to be hybridized, its GC nucleotide content, andany other parameter, for example following protocols described bySambrook et al, 2001 (Molecular Cloning: A Laboratory Manual, 3^(rd)Edition, Laboratory Press, Cold Spring Harbor, N.Y.).

In the above definition, and throughout the present text, the term“specific” should be taken to have its broadest meaning, normally usedin laboratories. Thus, a molecule A specifically recognizes a molecule Bif, in a complex mixture, molecule A has an affinity for molecule B thatis significantly higher than its affinity for other molecules of themixture, so that it is possible to detect molecule B via molecule A.

The stringency conditions used here are those that allow the PDIs ofdifferent Leishmania species to be detected rather than those of thehost and other microorganisms in the presence of a radiolabelled probesynthesized from the cDNA of LmPDI.

As an example, a probe of the invention, which specifically hybridizeswith sequence SEQ ID No: 2 under stringent conditions, is such that aSouthern blot carried out using said labeled probe, when carried out ona DNA sample from cells infected with a strain of L. major expressingLmPDI, has at least one clearly distinct band of higher intensity thanother bands (non specific), said band not appearing on a Southern blotcarried out under the same conditions on a DNA sample from cells notinfected by a strain of L. major.

In a further aspect, the invention concerns a nucleotide primer that canallow specific amplification of at least a portion of the sequence SEQID No: 1, from cells infected with Leishmania, thus allowing thepresence or absence of the virulence gene coding the LmPDI to bedetermined in a biological sample. Amplification will be termed“specific” if the amplification reaction carried out from control cellsnot infected with Leishmania does not result in significantamplification of any sequence, while the same reaction carried out on asample containing the nucleotide sequence of SEQ ID No: 1 results inamplification of at least one fragment of said sequence.

The probes and primers mentioned above can if necessary be labeledand/or presented in diagnostic kits which also form part of theinvention. It may be advantageous to determine the presence and possiblythe level of expression of the gene for LmPDI during an infection withLeishmania, for example to determine the parasitic and/or opportunisticcharge of a treatment involving the use of a LmPDI inhibitor.

In a further implementation, the invention provides purified antibodiesspecifically recognizing LmPDI. They may be monoclonal or polyclonalhuman, humanized or animal antibodies. Said antibodies can be purified,for example, on an LmPDI affinity column using the protocol described inthe experimental section. Said specific LmPDI antibodies may have anumber of applications.

They may serve to detect the presence of LmPDI in a biological sample,for example to diagnose leishmaniasis and/or to determine thepossibility of using a LmPDI inhibitor to treat that leishmaniasis.

Thus, the invention also concerns an in vitro method for diagnosing aninfection by a parasite responsible for leishmaniasis. Such a method canbe carried out using a polypeptide or a protein of the invention or anantibody directed against that protein, or using probes as definedabove.

A particular diagnostic method of the invention comprises the followingsteps:

-   -   bringing at least one antibody in accordance with the invention        into contact with a biological sample from a subject partially        infected by a parasite responsible for leishmaniasis under        conditions allowing the formation of an immune complex between        said antibody and antigenic proteins contained in the sample;    -   detecting said complex.

The complex can be detected using any means that is known to the skilledperson (enzymatic reaction, fluorescence transfer or the like).

The antibodies of the invention can be comprised in diagnostic kits inthe same manner as the probes or primers mentioned above.

Diagnostic kits for implementing the method described above form anintegral part of the present invention.

By way of example, such a kit can comprise

-   -   at least one antibody in accordance with the invention;    -   a medium suitable for forming an immune complex between the        antigenic proteins contained in the analyzed sample and said        antibody;    -   reagents allowing the detection of the complexes so formed;    -   if appropriate, control samples.

Alternatively, the antibodies of the invention can form part of thecomposition of a drug intended for prophylaxis, attenuation or for thetreatment of certain leishmaniases.

In a further aspect of the invention, the invention pertains to animmunogenic composition comprising a protein and/or a recombinantpolypeptide and/or a nucleic acid sequence and/or a vector and/or a cellof the invention as described above, said immunogenic composition beingcapable of in vitro stimulation of the proliferation of mononuclearcells deriving from individuals who have come into contact with aLeishmania parasite. A preferred immunogenic composition of theinvention is capable of in vitro stimulation of the proliferation ofmononuclear cells deriving from individuals who have come into contactwith Leishmania major.

In a preferred implementation of the immunogenic compositions of theinvention, said compositions have a formulation that is pharmaceuticallyacceptable for administration to a human or animal host.

The inventors have shown that LmPDI is susceptible of in vitro inductionof the production of cytokines by mononuclear cells deriving fromindividuals who have come into contact with L. major, and that theexpression profile of the cytokines corresponds to that observed duringa type Th1 immune response (Example 3). An immunogenic composition asdescribed above, which is capable of inducing a type Th1 immune responsewhen administered to a human or animal host, thus constitutes aparticularly preferred implementation of the present invention.

The invention also pertains to a vaccine composition comprising aprotein and/or a recombinant polypeptide and/or a nucleic acid sequenceand/or a vector and/or a cell of the invention as described above, saidvaccine composition being intended to protect a human or animal hostagainst leishmaniasis. Preferably, the vaccine compositions of theinvention are formulated in a manner that is pharmaceutically acceptablefor administration to a human or animal host.

Said vaccine composition can be in the liquid form for injection into apatient, either subcutaneously or intramuscularly, or in the form of anoral vaccine, in the form of a pomade, or in the form of particles boundto a nucleotide sequence of the invention, for example by DNA adsorptiononto the particle surface. This latter form allows the vaccine to beadministered using a gene gun. It is important to note that theformulations for the vaccine compositions mentioned here are givensolely by way of example and are in no way restrictive.

The immunogenic and/or vaccine compositions of the invention can alsocomprise one or more antigen(s) that are heterologous as regardsLeishmania, and/or one or more nucleic acid sequence(s) coding for saidantigens. The compositions of the invention can thus trigger animmunological reaction against several different pathogens and ifappropriate may constitute polyvaccines.

The vaccination process and the doses of active agent must be adapted tothe type of vaccine used and to the mammal to which it is administered.

Methods for vaccination against Leishmania, consisting of administeringa composition comprising a protein and/or a recombinant polypeptideand/or a nucleic acid sequence and/or a vector and/or a cell of theinvention as described above to a human or animal host, are alsoencompassed by the invention.

Determining the role of LmPDI in the virulence of Leishmania can alsoenable novel strategies for identifying active molecules for inhibitingthe growth of the parasite to be envisaged. It has been shown that amolecule inhibiting PDI, for example, such as bacitracin orchloromercuribenzene sulfonic acid (pCMBS), inhibits the growth ofLeishmania in a liquid medium (Example 4). Thus, the invention alsopertains to a method for screening molecules susceptible of inhibitingthe growth of Leishmania major, comprising a step for evaluating thecapacity of said molecules to inhibit the activity of LmPDI. Proteindisulfide-isomerases in general have a plurality of activities, inparticular oxido-reduction, isomerase, and chaperone activities. Thescreening methods of the invention can pertain to the inhibition of anyof the functions of LmPDI.

In a particular screening method of the invention, the step forevaluating the capacity of a molecule to inhibit the activity of LmPDIis carried out in a test for reactivating scrambled RNase A, comprisingthe following steps:

-   -   incubating scrambled RNase A in the presence of LmPDI under        conditions allowing its reactivation;    -   incubating scrambled RNase A under conditions identical to those        allowing its reactivation by LmPDI, the molecule to be tested        being added;    -   comparing the results obtained in the absence and in the        presence of the test molecule, a fault in the reactivation of        RNase A in the presence of the test molecule revealing that said        molecule has an LmPDI inhibiting activity.

Any other PDI activity test can be used in the screening methods of theinvention, in particular any test derived from the initial protocoldescribed by Lyles and Gilbert (1991).

A screening method of the invention can also comprise a test forinhibiting the growth of Leishmania major in a liquid medium and ifappropriate, a test for inhibiting the growth of Leishmania major in anexperimental murine model of leishmaniasis. An example of such a methodis described in the experimental section, Example 5.

The active molecules screened by the method defined above arecharacterized by their capacity to inhibit or modulate the growth ofLeishmania major.

The results obtained with bacitracin shown in Example 4 show that a PDIinhibitor can inhibit the growth of Leishmania The use of one or moreprotein disulfide-isomerase (PDI) inhibitors for the preparation of apharmaceutical composition intended for prophylaxis, attenuation, ortreatment of an infection with Leishmania thus forms an integral part ofthe invention. Compounds with an anti-PDI activity that can be used inaccordance with the invention that can be cited are anti-PDI oranti-LmPDI antibodies, bacitracin, zinc bacitracin,5,5′-dithiobis(2-nitrobenzoic) acid (DTNB), p-chloromercuribenzenesulfonic acid (PCMBS) or tocinoic acid.

The compositions prepared in accordance with the above uses canpreferably be topically, orally or parenterally administered to a humanor animal host.

In accordance with a particular aspect, the invention concerns the useof bacitracin or zinc bacitracin as an inhibitor to the growth of aparasite responsible for leishmaniasis or as an active agent against aLeishmania infection.

Clearly, a pharmaceutical composition for the treatment of an infectionwith Leishmania containing one or more protein disulfide-isomerase (PDI)inhibitors forms an integral part of the invention. Such a compositioncan in particular contain bacitracin or zinc bacitracin. The compositioncan be formulated for topical application, for example in the form of acream, an ointment, a pomade, or a spray, this list being non-limiting.The inventors have shown that such a composition, applied locally in theform of a pomade to the injection site of the parasite in BALB/c mice,attenuates the progress of the disease (Example 9 and FIG. 14).

The present invention also pertains to a pharmaceutical composition forthe treatment of an infection with Leishmania, comprising at least onespecific antibody for LmPDI and/or any molecule that inhibits PDIactivity. Such a composition is preferably appropriate for topical, oralor parenteral administration.

Methods for treating leishmaniases, comprising administration of a PDIor LmPDI inhibitor to a human or animal patient, whether an antibody orany other type of molecule, also fall within the scope of the invention.

The examples and figures below describe the biological experiments whichhave been carried out in the context of the present invention and whichprovide the required experimental support, without in any way limitingits scope. They also illustrate, in a non restrictive manner, certainaspects of the implementation and importance of the present invention.

KEY TO FIGURES

FIG. 1 shows a differential display (DD) analysis of the expression ofLeishmania major genes in the two most virulent isolates (94 and 67, V)and the two least virulent isolates (32 and 07, v).

FIG. 1A shows a portion of a sequencing gel after autoradiography,showing the products amplified by PCR using an arbitrary decamer and anoligo dT primer. The differentially expressed cDNAs are indicated byarrows. The p14 cDNA is indicated by an asterisk.

FIG. 1B shows a Northern blot analysis of the expression of a geneidentified by the DD technique between the most virulent isolates (94and 67, V) and the least virulent isolates (32 and 07, v). The mRNAextracted from the promastigotes from different isolates in thestationary growth phase were hybridized with the radiolabelled probep14. After autoradiography, the blots were de-hybridized thenre-hybridized with a specific probe for the gene for the α-tubuline ofL. Major (α-tub).

FIG. 2 shows the nucleotide sequence for the cDNA (SEQ ID No: 1) ofLmPDI and the deduced sequence of amino acids (SEQ ID No: 3). Thenucleotides in lower case letters represent non-translated regions. Theleader sequence (SL) of 18 nt is underlined and the potential sequencefor the polyadenylation signal is boxed. The potential sequence for thepeptide signal is shown in bold. The potential active sites for LmPDIare double underlined and the probable sequence for retention in theendoplasmic reticulum is shown as a broken line.

FIG. 3 shows the alignment of the amino acid sequence for LmPDI with theprotein disulfide-isomerase of Trypanosoma brucei (T. brucei, GenBankaccession no.: P12865), Hypocrea jecorina (H. Jecorina, 074568),Caemorhabditis elegans (C. elegans), 017908), Chiamydomonas reinhardtii(C. reinhard, 048949), Drosophila melanogaster (D. melano, P54399),Cryptosporidium parvum (C. parvum, Q27553), and Homo sapiens (H.sapiens, P072237). The letters boxed in black indicate identical aminoacids and those boxed in gray indicate similar amino acids. The “gaps”were introduced to obtain the maximum similarity between the alignedsequences and are indicated by dashes.

Two software programs were used to carry out the alignments:

-   -   CLUSTAL W version 1.8; Thompson, J D, Higgins, D G and Gibson, T        J;    -   BOXSHADE version 3.21; Hoffman, K and Baron, M.

FIG. 4 shows an analysis of the role of recombinant LmPDI inreactivating scrambled RNase. Scrambled RNase A (8 μM) was incubated ina buffer containing 4.5 mM (cCMP), 1 mM glutathione GSH, 0.2 mMglutathione disulfide GSSH, 2 mM EDTA and 100 mM Tris-HCl pH 8 in thepresence of bovine serum albumin (BSA) (1.4 μM) as a negative control,bovine protein disulfide-isomerase (1.4 μM) as the positive control, orrecombinant LmPDI (1.4 μM) for 30 minutes at 25° C. RNase A reactivationwas determined by measuring the RNase A activity at 296 nm every 5minutes for 30 minutes (Lyles and Gilbert, 1991).

FIG. 5A shows a Southern Blot analysis for the number of copies of theLmPDI gene in the Leishmania major gene. 8 μg of isolate genomic 94 DNAfrom L. major was digested by the following restriction enzymes: AvaI,EcoRV, HindIII, PstI, EcoRI, XhoI, NcoI, SacI, SphI. The enzymes markedwith an asterisk cleave once within the cDNA of LmPDI.

FIG. 5B shows a Southern Blot analysis of the LmPDI gene in differentspecies of Leishmania. 8 μg of genomic L. major DNA (94), dermotropic L.infantum (L. infantum MC), viscerotropic L. infantum (L. infantum Visc);L. donovani were digested with the PstI enzyme. Genomic DNA washybridized in these experiments by the probe representing the entirecDNA sequence of LmPDI.

FIG. 6 shows immunodetection of native LmPDI in L. major with differentpreparations of anti-LmPDI antibodies. 20 μg of total promastigote GLC94 proteins in Laemmli buffer (track 1) or in the presence of 0.5 m ofDTT (track 2) and 0.05 μg of LmPDI produced in E. coli bacteria andpurified (rLmPDI) (track 3) underwent electrophoresis then weretransferred onto a nitrocellulose membrane, then revealed with ananti-LmPDI immunoserum (track 1) or anti-LmPDI antibodies purified on anaffinity column (tracks 2 and 3).

FIG. 7 shows a Western blot analysis of the expression of LmPDI in thetwo most virulent isolates (94, 67, V) and the two least virulentisolates. (32, 7, v) from promastigotes. 20 μg of total promastigoteprotein in the stationary growth phase from different isolates underwentelectrophoresis and were then transferred onto a nitrocellulose membranewhich was incubated in the presence of polyclonal anti-LmPDI antibody.The arrows (>) indicate the 3 proteins recognized by the anti-LmPDIimmunoserum.

FIG. 8 shows the proliferation of mononuclear cells from individualsliving in a zoonotic cutaneous leishmaniasis region in Tunisia, afterincubation of LmPDI (5 μg/ml). The lymphomonocytary cells wererecovered, washed by 3 successive centrifuge runs with RPMI-PS/Glumedium (30 ml then twice 10 ml) then counted and incubated at aconcentration of 10⁶ cells/ml of medium in the presence or absence of aconcentration of 5 μg/ml of LmPDI. After 5 days of culture, lymphocytestimulation was estimated by incorporating tritiated thymidine. Theresult is expressed in CPM.

FIG. 9 shows the results of a parasite (L. major) growth inhibition testin a liquid medium using bacitracin. They are growth curves taken over96 hours, for promastigotes of L major in the presence of 0, 1 mM, 1.5mM or 2 mM of bacitracin.

FIG. 10 shows the effect of bacitracin (BAC), zinc bacitracin (BACZn),p-chloromercuribenzoic acid (PCMBA) and tocinoic acid (TOC) on the invitro activity of recombinant LmPDI. Different concentrations ofinhibitors (0 to 2 mM) were used to follow the effect of PDI inhibitorson the capacity of LmPDI to reactivate scrambled RNase A in vitro. LmPDIwithout inhibitors was used as the positive control (T).

FIG. 11 shows the effect of bacitracin (BAC) (FIG. 11A), zinc bacitracin(BACZn) (FIG. 11B), 5,5′dithiobis-(2-nitrobenzoic acid) (DTNMB) (FIG.11C) and p-chloromercuribenzoic acid (PCMBA) (FIG. 11D) on the in vitrogrowth of Leishmanias in a liquid medium. Different concentrations ofinhibitors (0 to 5 mM) were used to follow the effect of PDI inhibitorson the multiplication of parasites in vitro. Parasites that had not beentreated with inhibitors (T) were selected as a control for theseexperiments.

FIG. 12 shows the inhibition of the activity of rLmPDI by bacitracin andzinc bacitracin. The effect of bacitracin (BAC) and zinc bacitracin(BACZn) on the activity of rLmPDI was measured in vitro. Differentconcentrations of BAC and BACZn inhibitors (0 to 2 mM) were tested toanalyze their effect on the capacity of rLmPDI to reactivate scrambledRNase in vitro. The activity of rLmPDI in the absence of inhibitorsacted as a positive control.

FIG. 13 illustrates the inhibition of the multiplication of GLC94promastigotes by zinc bacitracin. The effect of zinc bacitracin (BACZn)on the multiplication of GLC94 promastogotes was determined in vitro.Different concentrations of inhibitor were tested to analyze theireffect on the capacity of the parasites to multiply in vitro. Thecontrol (C) was constituted by parasites cultivated in a complete mediumin the absence of inhibitors.

FIG. 14 shows the effect of zinc bacitracin on the evolution of thedisease in sensitive BALB/c mice infected with GLC94 isolatepromastigotes. Sensitive BALB/c mice were infected with 10⁶promastigotes from the GLC94 isolate into the plantar pad and treated ornot treated with bacitracin. The treatment was halted 9 weeks afterinfection (the arrow indicates treatment stoppage). Each curve shows thechange in the size of a single mouse.

EXAMPLES

The experimental results shown in the following examples were obtainedusing the following materials and methods:

Parasites and Culture Conditions

The L. major isolates used in this study derived from human ZCL lesionsobtained during the study summarized in Example 1. The parasites werecultivated in NNN medium (solid medium prepared and based on agarose andrabbit blood) at 26° C., and progressively transferred into RPMI (SIGMA,St Louis, Mo.) containing 2 mM of L-glutamine, 100 U/ml of penicillin,100 μg/ml of streptomycin and 10% of deactivated fetal calf serum(complete medium). Promastigotes in the logarithmic growth phase wereadjusted to 10⁶ parasites/ml in a constant volume of complete medium andincubated at 26° C. The stationary growth phase was reached after 4 to 6days with the density of parasites of 3×10⁷ to 8×10⁷ parasites. Thosepromastigotes in the stationary growth phase were used for RNA andprotein extractions.

RNA Extraction and Differential Display

Total RNA was extracted using the “TRIZOL” reagent (Gibco-BRL). PolyA⁺RNA was purified by passage through an oligo dT/cellulose column using a“poly A⁺ RNA isolation kit” (Amersham-Pharmacia) following themanufacturer's instructions. 200 ng of mRNA was used in a reversetranscription reaction of 20 μl containing 1 μM of an oligo(dT)₁₁MNprimer, with M=A or C or G and N=A or C or G or T (Genset), 1×FirstStrand Buffer (Gibco-BRL), 5 μM dNTP (Amersham-Pharmacia), 10 U ofRNAsin (Promega) and 200U of reverse transcriptase (Gibco-BRL).

After incubating at 37° C. for one hour, the reaction was stopped byincubating for 5 minutes at 95° C. The cDNA was amplified by PCR using acombination of 12 oligo dT and 10 arbitrary decamers. PCR was carriedout in a volume of 20 μl containing 2 μl of reverse transcriptionreaction, 0.2 μM of 5′ primer, 1 μM of 3′ primer, 2 μM of dNTP, 10 μCi[α³⁵S] of ATP, 1×Taq DNA polymerase reaction buffer and 1U of Taqpolymerase (Amersham-Pharmacia). The reactions were incubated in aPerkin-Elmer 9600 thermocycler for 40 cycles at 94° C. for 30s, 40° C.for 60s and 72° C. for 30s followed by one cycle at 72° C. for 6minutes. The PCR products were analyzed on a 6% acrylamide sequencinggel. The gel was vacuum dried on Whatmann 3MM paper andautoradiographed. The differentially expressed cDNA was excised from thegel, eluted and reamplified by PCR in the presence of the sameoligonucleotides, under the conditions described above. Theamplification products were cloned in pMOSblue vector using theBlunt-ended PCR cloning kit (Amersham Pharmacia), following themanufacturer's instructions. The cloned fragments were sequenced using aSequencing Ready Reaction Kit (Perkin-Elmer) and analyzed using the ABI377 automatic sequencer.

Northern Blot Analysis

200 ng of mRNA from promastigotes extracted during the stationary growthphase of 4 isolates from L. major were denatured, separated on a 1.2%agarose/2.2 M formaldehyde gel and transferred by capillarity on a“Hybond N⁺” (Amersham-Pharmacia) membrane. The nucleic acids were thenfixed by heating for 2 hours at 80° C. The differentially expressed cDNAfragments and α-tubulin were labelled with [α³²P]dCTP using theMegaprime DNA labelling system kit (Amersham-Pharmacia). Hybridizationswere carried out in a 1× Denhardt's/6×SSC/0.1% SDS/0.1 mg.ml⁻¹ salmonsperm solution overnight at 65° C. The membranes were washed at 65° C.in a solution containing 0.1×SSC/0.1% SDS and autoradiographed.

Construction of a cDNA Library and Characterization of LmPDI cDNA

A cDNA library was constructed from 5 μg of mRNA from promastigotes fromthe most virulent strain (GLC94) in the ZAPII vector, following themanufacturer's instructions (Stratagene). 6×10⁶ lysis plaques werescreened using the p14 probe labeled with [α³²P] dCTP using theMegaprime DNA labelling system kit (Amersham-Pharmacia). The lysisplaques of interest were removed and screened again to isolate positiveclones from contaminating clones. The positive clones were thensequenced.

Southern Blot Analysis

10 μg of genomic DNA extracted from promastigotes from the most virulentstrain GLC94 were digested with the restriction enzymes indicated inFIG. 5 and analyzed on a 0.6% agarose gel, then transferred to a HybondN⁺ (Amersham-Pharmacia) membrane. The membrane was incubated in thepresence of a probe radioactively labeled with [α³²P]dCTP andcorresponding to the entire cDNA clone of LmPDI. The membranes were thenwashed in a solution containing 0.1×SSC/0.1% SDS and autoradiographed.

Expression and Purification of the Recombinant Protein LmPDI in E. coliBL21 Bacteria

The sequence corresponding to the open reading frame of cDNA of LmPDI(1371 bp) deprived of the sequence coding for the peptide signal wascloned in the bacterial expression vector pET-22b (Novagen). E. coliBL21 bacteria containing the recombinant plasmid (pET-22b-LmPDI) werecultivated in LB medium then synthesis of the recombinant protein wasinduced in the presence of 1 mM of isopropyl-1-thio-D-galactopyranoside(IPTG) for 4 hours. The recombinant protein LmPDI-(His)₆ (SEQ ID No: 4)was purified by affinity chromatography on a nickel column (Ni²⁺)(Amersham-Pharmacia). The purity of the protein produced was verified bySDS-PAGE.

Production of a Polyclonal Anti-LmPDI Antibody and Analysis ofExpression of the Native Protein by Immunoblot

A rabbit was immunized by intramuscular injection of 500 μg ofemulsified purified recombinant LmPDI in incomplete Freund's adjuvant(IFA, Sigma) (v/v). The rabbit received two additional injections of 500μg of recombinant protein, the first intramuscularly 15 days after thefirst injection and the second intradermally 30 days later. The rabbitwas bled 10 days after the last injection; the serum was harvested andkept at −80° C. The protein lysate from the promastigotes was denaturedin Laemmli 1× buffer for 10 minutes at 100° C., deposited on a 12%SDS-acrylamide gel and electrotransferred onto a nitrocellulose membrane(Millipore). The membranes were incubated in a saturated PBS/0.1%Tween20/3% skimmed milk solution at ambient temperature for one hour,then in the same solution containing anti-LmPDI antibody diluted to{fraction (1/1000)}^(th) at 4° C. overnight. After 3 washes in PBS/0.1%Tween20, the membranes were incubated in the presence of secondaryrabbit anti-IgG antibody coupled with peroxidase (Amersham-Pharmacia,diluted to {fraction (1/1000)}) for one hour at ambient temperature andwashed 3 times in PBS/0.1% Tween20. The protein-antibody complexes wererevealed by detecting the peroxidase activity using the “ECL system”kit, following the manufacturer's instructions (Amersham-Pharmacia).

Preparation of Scrambled RNase A

20 mg of purified ribonuclease (RNase A) was scrambled at ambienttemperature for 18 hours in a buffer containing 0.15 M of DTT, 6 M ofguanidine-HCl and 0.1 M of Tris-HCl at a pH of 8.6 before being purifiedon a Sephadex G-25 column equilibrated in 0.01 M HCl. The concentrationof scrambled RNase A fractions was determined using an extinctioncoefficient of 9200 M⁻¹ cm⁻¹ at 275 nm. The fractions were stored at−80° C. for two weeks.

Reactivating RNase A in the Presence of Recombinant LmPDI Protein

Scrambled RNase A (8 μM) was incubated in a buffer containing 4.5 mMcCMP, 1 mM glutathione GSH, 0.2 mM glutathione disulfide GSSH, 2 mM EDTAand 100 mM of Tris-HCl pH 8 in the presence of bovine serum albumin(BSA) (1.4 μM) as a negative control, bovine protein disulfide-isomerase(1.4 μM) as a positive control, or recombinant LmPDI (1.4 μM) for 30minutes at 25° C. RNase A reactivation was determined by measuring theRNase A activity at 296=m every 5 minutes, as described in theliterature (Lyles and Gilbert, 1991).

Example 1 Selection of L. major Isolates Having Different Levels ofVirulence

The L. major isolates used in this study derived from human ZCL lesionsobtained during a prospective study carried out in 1994-1995 at ElGuettar, in southern Tunisia (Louzir, Melby et al, 1998). They had beenselected from 19 isolates on the basis of their pathogenic power duringexperimental infection of sensitive BALB/c mice: 2×10⁶ amastigotes fromvarious isolates were injected into the rear paw pads of BALB/c mice andthe progress of the lesion was observed every week for 9 weeks. Fiveweeks after infection, the production of IL-4 and IFN-γ by mononuclearcells of lymphatic ganglia activated in vitro by antigens from theparasite was measured.

These experiments showed firstly the great heterogeneity in the progressof the disease induced by different isolates of L. major and secondly,that using a single isolate leads to reproducible results.

The most virulent strains induced the greatest amount of IL-4 and thelowest levels of IFN-γ in vitro, 5 weeks after infection.

From the observation that clinical expression of infection with L. majorvaries depending on the strains and is reproducible within each of themin the experimental model of infection of BALB/c sensitive mice, theinventors devised the hypothesis that the genes involved in virulencecould be differentially expressed when comparing the most virulentisolates with the least virulent isolates. A preliminary analysis of theexpression of a group of genes already described by other authors andassociated with the virulence of the parasite, including LPG1, LPG2,KMP-11, Cpc, Cpb, Hsp100, Gene B and gp63, was carried out by a reversetranscription technique and quantitative gene amplification. Thisanalysis did not show any difference between L. major isolatesexpressing a different pathogenicity in BALB/c mice (Kebaier, Louzir etal, 2001).

Two isolates, MHOM/TN/94/GLC94 and MHOM/TN/94/GLC67 (GLC94 and GLC67respectively), which induced severe lesions, developed rapidly andrepresented the most virulent isolates, and 2 isolates MHOM/TN/94/GLC07and MHOM/94/GLC32 (GLC07 and GLC32 respectively) inducing a less severeexperimental disease and representing less virulent isolates, wereselected to continue the search for virulence genes potentiallyexpressed at different levels depending on the strains.

Example 2 Differential Display Identification of a Novel ProteinDisulfide Isomerase LmPDI of Leishmania major, Involved in NaturalParasite Virulence

1-Identification of Genes Differentially Expressed in Virulent Isolatesand Low Virulence Isolates of L. major

mRNA was firstly purified from promastigotes from two highly virulentisolates (GLC94 and GLC67) and from two low virulence isolates (GLC32and 07) then reverse transcribed to cDNA using Oligo(dT)₁₁MN primers,where M=A or C or G and N=A or C or G or T. The primer used during thedifferential display experiments was:

The amplification reactions were carried out by PCR using the sameoligo-dT primers used during the reverse transcription reaction andcombined with 10 arbitrary primers, as described in the scientificliterature (Liang and Pardee, 1992; Liang, Bauer et al, 1995; Heard,Lewis et al, 1996).

In total, 60 combinations of primers were produced and analyzed.Polyacrylamide gel analysis of the amplification products usingdifferent combinations of primers showed that the genes from differentisolates from L. major(highly virulent or low virulence) express, inapproximately 95% of cases, the same mRNA and at equivalent levels.Taken alone, 25 messengers appear to be differentially expressed betweenhighly virulent and low virulence isolates (FIG. 1A). The differentiallyexpressed cDNA was firstly isolated from the acrylamide gel thenreamplified using the same combinations of primer used during the firstPCR and finally cloned in pMOS vector. Sequencing the different clonesshowed that a certain number of them were identified.

Analysis of the mRNA from the different isolates of L. major by NorthernBlot using the 14 fragments differentially expressed as a probe showedthat 3 clones out of the 14 isolates exhibit differential expressionbetween highly virulent isolates and low virulence isolates. One ofthese clones, p14, has been characterized by Northern Blot. The probecorresponding to clone p14 specifically hybridized with a transcriptwith an approximate size of 2.2 kb, which is preferentially expressed inthe two most virulent isolates compared with the two least virulentisolates (FIG. 1B). This confirms the results obtained by theDifferential Display technique. Clone p14 has been completely sequencedand the size of this clone is 339 bp. A comparison of the nucleotidesequence of this fragment with the sequences described in the databases(GenBank and EMBL) did not identify a significantly homologous sequence.This could be due to the fact that the p14 clone corresponds to the nontranslated 3′ terminal region of the messenger.

2—Cloning and Analysis of the Entire cDNA p14 Sequence

To isolate the entire cDNA sequence corresponding to the p14 clone, the339 bp fragment was used to screen a cDNA bank of promastigotes of theGLC94 isolate. Two positive clones were isolated from 6×10⁵ recombinantclones analyzed. FIG. 2 shows the nucleotide sequence of the longestclone, which is 2094 bp (SEQ ID No: 1). This clone has an open readingframe coding for a 477 amino acid (aa) polypeptide with a theoreticalmolecular weight of 52.4 kDa and an isoelectric point of 5.22. TheN-terminal region of this protein corresponds to a potential peptidesignal for export to the endoplasmic reticulum, 20 aa long. The nontranslated 5′ region contains a splice leader sequence characteristic ofLeishmanias and the non translated 3′ region contains a poly A tailpreceded by a potential polyadenylation site (FIG. 2).

The peptide sequence for the isolated clone showed 27-36% identity withproteins of the protein disulfide isomerase family (PDI and Erp) ofseveral species (FIG. 3). Further, this protein contains two regions atresidues 47-52 and 381-386 which are identical to the potential activesites (Cys-Gly-His-Cys, or CGHC) of PDI, Erp and proteins from thethioredoxin family. The C-terminal portion shows a potential signal forretention in the endoplasmic reticulum of the KDEL (EEDL) type atresidues 474-477 suggesting that, like PDI and Erp, this protein isfound in the cavity of the endoplasmic reticulum. P14 is thus a proteinfrom the L. major protein disulfide isomerase family. It has beendenoted LmPDI (FIGS. 2 and 3).

To determine whether LmPDI is endowed with an oxido-reductasethiodisulfide activity as demonstrated for the majority of the proteindisulfide-isomerase described, the capacity of the recombinant proteinLmPDI to renature denatured RNase A was studied. Recombinant LmPDIprotein was synthesized in E coli than purified and used in a test, invitro, for reactivating RNase. The results obtained show that LmPDI iscapable of restoring RNase A activity in a similar manner to that ofbovine PDI, used as a control (FIG. 4).

To identify the number of copies of the gene coding for LmPDI, theinventors carried out Southern Blot type hybridization using as a probethe cDNA fragment of ³²P labeled LmPDI. The results obtained generallyshowed a single band, except for enzymes with a cleavage site within thecDNA of LmPDI (FIG. 5A). The gene coding for LmPDI is thus probablypresent in a single copy in the genome for L. major. Further, the genefor LmPDI appears to be conserved in different species of the Leishmaniatested (Leishmania infantum, dermotropic, and a viscerotrope, Leishmaniadonovani) FIG. 5B).

3-Immunoblot Analysis of LmPDI Expression

To characterize the expression of the native protein, a rabbit wasimmunized with recombinant LmPDI protein synthesized in E coli andpurified by affinity chromatography. Using immunoblot, the inventorshave shown that the anti-LmPDI polyclonal antibody obtained stronglyrecognized a protein of the expected size (55 kDa) in lysates frompromastigotes in the stationary growth phase of GLC94 (FIG. 6). Further,two other proteins were detected. The first had a molecular weight of105 kDa, corresponding to about twice that of LmPDI, and the second hada molecular weight of 35 kDa. In order to verify whether the 105 kDaprotein corresponded to a dimer of LmPDI, denatured GLC94 promastigotelysates were analyzed in the presence of high concentrations of DTT (0.5mM). Under these conditions, the anti-LmPDI detected no more proteins of105 kDa. These results suggest that LmPDI is organized into oligomers.The 35 kDa protein appears to be a contaminant. In fact, anti-LmPDIpurified on an affinity column (Sepharose 4B-LmPDI) no longer recognizesthe 35 kDa protein (FIG. 6).

In order to compare the level of expression of LmPDI between the mostand the least virulent isolates, promastigote proteins were extractedthen quantified in the stationary growth phase. 5 μg of proteins wereanalyzed on a 12% polyacrylamide-SDS gel and transferred to anitrocellulose membrane. Western blot analysis using arm anti-LmPDIantibody showed that LmPDI (55 kDa) and its dimer (105 kDa) were morestrongly expressed in the most virulent isolates (FIG. 7). In contrast,the 35 kDa contaminating protein was expressed in an equivalent mannerregardless of the test strain. These results suggest a correlationbetween the level of expression of LmPDI and the pathogenic power of thestudied isolates.

Example 3 Induction by LmPDI of In Vitro Proliferation of MononuclearCells from Individuals Having Active Lesions or ZCL Antecedents

L. major LmPDI, because of its high expression during the infectiousstage of the parasite, could be a target for a cellular immune response.In order to verify the pertinence of this hypothesis, the capacity ofLmPDI to induce a cellular immune response was evaluated by means ofexperiments on the proliferation of mononuclear cells obtained fromindividuals having active lesions or ZCL antecedents.

This study was carried out in 37 individuals living at El Guettar(southern Tunisia) for whom the results of the cellular proliferationtest against total antigens from the parasite (SLA, a test indicatingprior contact with the parasite) was available. These individuals weredivided up as follows:

-   -   Group 1: composed of 8 individuals with a negative SLA test;    -   Group 2: composed of 29 individuals with a positive SLA test.

Mononuclear cells comprising lymphocytes and monocytes were separatedfrom peripheral blood by centrifuging on a Ficoll/Hypaque gradient(Pharmacia, Uppsala, Sweden).

The results (FIG. 8) show significant proliferation with immuneindividuals.

Cytokines (IFN-β, IL-4) in PBMC culture supernatants were induced byincubating mononuclear cells for 48 hours with the same concentration ofLmPDI and assaying by means of an ELISA test using human anti-IL-4 andanti-IFN-β monoclonal antibodies (Pharmingen, San Diego, Calif.).

The results came from a small sample of individuals. They clearly showthe absence of IL-4 and the presence of significant amounts of IFN-β inthe supernatant from cells stimulated by LmPDI.

This result shows an essentially type Th1 response, indicating thatLmPDI could constitute a vaccine candidate against Leishmania.

Example 4 Inhibition of Growth of Leishmania major in a Liquid Medium inthe Presence of a PDI Inhibitor

Bacitracin is a known PDI inhibitor. Experiments using bacitracin showedthat at the final concentration of 2 mM, bacitracin completely inhibitedthe growth of L. major parasites in a liquid medium (FIG. 9).

These experiments were carried out under the following experimentalconditions:

a) Preparation of scrambled RNase A:

20 mg of purified ribonuclease (RNase A) was reduced and denatured atambient temperature for 18 hours in a buffer containing 0.15 M of DTT,6M of guanidine-HCl and 0.1 M of Tris-HCl at a pH of 8.6, before beingpurified on a Sephadex G25 column equilibrated in 0.01 M HCl. Theconcentration of scrambled RNase A fractions was determined with thehelp of an extinction coefficient of 9200 M⁻¹ cm⁻¹ at 275 μm. Thefractions were stored at −80° C. for two weeks.

b) Reactivation of RNase A in the Presence of Recombinant LmPDI Protein

The scrambled RNase A (8 μM) was incubated in a buffer containing 4.5 mMcCMP, 1 mM glutathione GSH, 0.2 mM glutathione disulfide GSSH, 2 mM EDTAand 100 mM Tris-HCl, pH 8, in the presence of bovine serum albumin (BSA)(1.4 μM) as a negative control, bovine protein disulfide-isomerase (1.4μM) as a positive control, or recombinant LmPDI (1.4 μM) for 30 minutesat 25° C. RNase A reactivation was determined by measuring the RNase Aactivity at 296 nm every 5 minutes for 30 minutes as described in theliterature (Lyles and Gilbert, 1991).

c) In Vitro Tests for Inhibition of the Thiodisulfide Oxido-ReductaseActivity of Recombinant LmPDI by Different PDI Inhibitors

The experimental conditions for the thio-disulfide oxido-reductaseactivity inhibition tests for recombinant LmPDI were strictly identicalto those described in the paragraph “Reactivation of RNase A in thepresence of recombinant LmPDI protein”, except that the reactions werecarried out in the presence of 0.01 mM, 0.1 mM, 0.5 mM and 2 mM of thefollowing PDI inhibitors:

-   -   bacitracin;    -   zinc bacitracin;    -   p-chloromercuribenzoic acid (pCMBA);    -   tocinoic acid.        d) Inhibition of Parasite (L. major) Growth in a Liquid Medium

With the aim of determining the effect of bacitracin (BAC), zincbacitracin (BACZn), p-chloromercuribenzoic acid (pCMBA) and5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) on the in vitro growth ofLeishmanias in a liquid medium, different concentrations of theinhibitors cited above, 0 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 1.5mM, 2 mM, 2.5 mM and 5 mM were added to RPMI supplemented with 5% fetalcalf serum and containing 2×10⁶/ml of parasites in the exponentialgrowth phase. The parasites were incubated at 26° C. and counted every24 hours over 96 hours. The parasites were counted on Mallassez cells.

Example 5 Evaluation of LmPDI Inhibiting Molecules as Regards theirCapacities to Stop the Growth of Leishmania major

The evaluation of the role of LmPDI in Leishmania virulence allows novelstrategies for identifying molecules that are active in treatingLeishmania to be envisaged. It is probable that molecules known fortheir anti-PDI activities other than bacitracin are capable ofinhibiting the growth of the parasite. An example of a protocol forevaluating molecules potentially effective against Leishmania ispresented here.

An evaluation of molecules known for their anti-PDI activities or thosewhich will be newly identified can be carried out in three steps. Duringthe first step, the molecules are tested in vitro on recombinant LmPDIprotein produced in Escherichia (E.) coli. Tests for inhibiting thegrowth of parasites in a liquid medium are then carried out and finally,the molecules are tested in the experimental murine model ofleishmaniasis.

1—Evaluation of the Inhibition of Recombinant LmPDI.

The detailed technique for analyzing the PDI activity of LmPDI wasdescribed in Example 2 and in the Materials and Methods section above.The same technique can be used to evaluate the capacity of certain PDIinhibitors which are known or still to be identified (using a molecularmodel of LmPDI), by adding different concentrations of potentialinhibitors to the reaction volume. The results are expressed as thepercentage inhibition compared with the buffer alone. Only moleculeswith a significant and dose-dependent LmPDI inhibiting activity areretained.

With the aim of determining whether the different PDI inhibitorsdescribed in the literature could inhibit the thio-disulfideoxido-reductase activity of LmPDI, the capacity of these inhibitors toblock the enzymatic activity of LmPDI synthesized in E. coli thenpurified was studied in an in vitro test, at different concentrations.The inhibitors were:

-   -   bacitracin;    -   zinc bacitracin;    -   p-chloromercuribenzoic acid (pCMBA);    -   tocinoic acid.

The results obtained are shown in FIG. 10 and show that the LmPDIactivity is completely inhibited in the presence of 0.01 mM of pCMBA and2 mM of bacitracin or zinc bacitracin. In contrast, tocinoic acid didnot appear to have a very great effect on the activity of LmPDI at theconcentrations employed (concentrations which completely inhibit theactivity of human PDI).

2—Inhibition of the growth of parasites (L. major) in a liquid medium.The test molecule was dissolved in a solvent the suitability of whichdepended on its physico-chemical properties (solubility in aqueoussolutions or organic solvents). In all cases, the solvent alone was usedas a control. As an example, the experiments could be carried out on theGLC94 L. major isolate. The composition of the culture medium was givenabove (Example 2 and Materials and Methods). The cultures were incubatedat 26° C. and re-pricked out regularly to maintain the parasites in thestationary growth phase. For certain experiments, the amastigote-likestage of the parasite was used. In this case, the parasites(promastigotes) of the stationary growth phase were centrifuged, themedium was replaced with Schneider Drosophila medium adjusted to a pH of5.0 and supplemented with fetal calf serum (FCS). The cultures were thenincubated under 5% CO₂ at 35° C.

Inhibition of the growth of L. major promastigotes was carried out onparasites taken in the exponential growth phase, adjusted to the initialconcentration of 10⁶ parasites/ml of complete medium and incubated in anamount of 100 μl/well in 96-well culture plates in the absence orpresence of different concentrations of the test molecule. The parasiteswere incubated in 5% CO₂ at 26° C. and counted every 24 h for 96h.Parasite counting was carried out on a Mallassez cell. Alternatively, ahemocytometer could be used. All of the measurements were carried out intriplicate. The inhibiting capacity of a molecule was determined as theinhibiting concentration which reduces cell division by 50% comparedwith the control (IC50).

The reduction in the viability of amastigotes was evaluated using afluorimetric test employing Almar Blue as a viability/growth indicator.

The PDI inhibitors described in paragraph (1-) were tested with the aimof evaluating their capacities to inhibit the in vitro growth ofparasites. To this end, different quantities of inhibitors were added toRPMI containing 2×10⁶/ml of parasites in the exponential growth phase.The parasites were incubated at 26° C. and counted every 24 hours over96 hours. The parasites were counted on Mallassez cells. The resultsobtained are shown in FIG. 11 and show that bacitracin, zinc bacitracinor pCMBA completely inhibited the growth of leishmania in concentrationsof 5 mM and 2 mM and 0.5 mM respectively. In contrast,5,5′-dithiobis(2)nitrobenzoic acid) (DTNB) in the concentrations used(concentrations which completely inhibit the activity of human PDI) didnot appear to have a very large effect on the growth of Leishmanias.

3—Evaluation of the efficacy of pre-selected inhibitors in theexperimental model of infection of sensitive BALB/c mice by L. major.The in vivo experiment will depend on the toxicity and physico-chemicalproperties of the test molecules. BALB/c mice will be infected by 10⁶ L.major promastigotes obtained during the stationary growth phase andinjected (in a volume of 50 μl) into the plantar pad of the rear rightpaw. The lesion diameter will be measured weekly using sliding calipers.

In all, three therapeutic protocols will be applied depending on thecase:

-   -   for hydrophobic molecules, which diffuse well, and are slightly        toxic or non-toxic, the product will be injected        intraperitoneally at different concentrations and using        different schemes. The frequency of injection will depend on the        bioavailability of the molecule and on its half-life. In all        cases, the protocol will be stopped at the end of 9 weeks        following infection;    -   for hydrosoluble and relatively toxic molecules, the injections        will be made intra-lesionally (in general, the active doses can        be divided by 10) by dint of at least four injections into the        indurated zone;    -   for liposoluble molecules, a pomade will be tested by weekly        application to the experimental lesion.

Overall, and regardless of the mode of injecting the test product, twotypes of protocols will be carried out:

-   -   a protocol which starts immediately after injecting the        parasites;    -   a protocol which starts 4 to 5 weeks after injecting parasites,        at a time at which the lesion will already have been        established.

In all cases, at the end of the protocol, the mice will be sacrificedand an estimate of the parasitic load will be made at the injection siteand in the ganglion which drains the lesion.

Example 6 In Vitro Infection for Murine Macrophages by Leishmania

Murine bone marrow macrophages (MBMM) were obtained from bone marrowextruded from a femur or tibia from female BALB/c mice. The MBMM wascultivated in multi-chamber plates in an amount of 1.5×10³ cells perwell in 500 μl of complete medium. To stimulate the growth andmaturation of the MBMM, the culture medium was supplemented with 20% ofmedium conditioned with L-929 fibroblasts as a source of macrophagecolony stimulating factor (MCSF). After 6 days of culture at 37° C. and5% CO₂, the medium was removed, the MBMM was washed, and fresh RPMImedium with 10% fetal calf serum but comprising no medium conditioned byL-929 fibroblasts was added. Intra-lesional amastigotes were purifiedfrom non-ulcerated lesions by differential centrifugation and countedusing trypan blue viral stain. These parasites were used to infect theMBMM in a final ratio of four parasites per macrophage. Two hours afteradding the parasites, the macrophages were washed five times with PBS toeliminate non phagocytary amastigotes. The cultures were then incubatedat 37° C. in 95% air and 5% CO₂. The experiments were carried out atdifferent points in time: 30 minutes and 2, 24 and 72 hours. At theindicated times, the wells were rinsed with PBS, the covers were removedand the infected macrophages were fixed with ethanol for 1 hour atambient temperature. The plates were then washed and stained with Giemsato follow the infection.

The infected macrophages were counted in the centre of each well werethe cells were well spread out and the parasites could be countedeasily. At this level of the plate, the parasite/macrophage ratio couldbe more than 4.

Example 7 Inhibition of the Enzymatic Activity of Recombinant LmPDI byProtein Disulfide-Isomerase Inhibitors

Several protein disulfide-isomerase (PDI) inhibitors have been describedin the literature (Ryser et al, 1994, Orlandi 1997, Mou et al, 1998). Ofthese, bacitracin and zinc bacitracin constitute a complex ofpolypeptide antibiotics produced by Bacillus subtilis and Bacilluslichenformis. Bacitracin A is the principal compound of commercialbacitracin, which is a mixture of at least nine bacitracins. Thisantibiotic is capable of inhibiting synthesis of the wall of manyGram+bacteria, but also the activity of many proteases such as PDI,transglutaminase, papain and neuropeptidase. The majority of thoseproteases have a cysteine residue in their active site.

In a first step, the investors tested the effect of these inhibitors inverifying their possible ability to alter the enzymatic activity ofrecombinant LmPDI (rLmPDI) in vitro.

The scrambled RNase technique described above (Lyles and Gilbert, 1991)was used to demonstrate the activity of LmPDI. Twenty milligrams ofRNase A (Amersham-Pharmacia) was denatured in a buffer composed of 0.15M dithiothreitol, 6M guanidine HCl and 0.1 M Tris-HCl, pH 8.6 for 18hours at ambient temperature. The scrambled RNase was then purified on aSephadex G25 column equilibrated in HCl 0.01 M and quantified byspectrophotometry at 275 nm.

In a glutathione-based reducing buffer, PDI catalyzes renaturing ofscrambled RNase (Gilbert, 1998). Restoration of RNase activity wasmeasured by spectrophotometry in the presence of cytidine 2′-3′-cyclicmonophosphate (cCMP) as a substrate. 8 μM of scrambled RNase, alone orin the presence of 1.4 μM of bovine serum albumin (BSA) or 1.4 μM ofrLmPDI was mixed in a buffer containing 4.5 mM of cCMP, 1 mM of reducedglutathione (GSH), 200 μM of oxidized glutathione (GSSG), 2 mM EDTA and100 mM Tris-Cl, pH 8. The reaction was carried out at ambienttemperature for 30 minutes. The hydrolysis of cCMP resulting from therenaturing of RNase was recorded by measuring the absorbance at 296 nmevery 5 minutes for the half hour of the reaction.

The activity of the recombinant LmPDI (rLmPDI) was measured in thepresence of different concentrations of bacitracin (BAC 0.01 mM-2 mM)and zinc bacitracin (BACZn, 0.01 mM-2 mM). The results are shown in FIG.12.

These results show that bacitracin and zinc bacitracin have similareffects. In the presence of these two products, 50% inhibition wasobserved at 0.1 mM, 70% at 0.5 mM and 100% at 2 mM. The concentrationswhich inhibit rLmPDI are comparable with those described in theliterature as inhibitors for PDIs from other species.

Example 8 In Vitro Growth Kinetics of L. major Promastigotes in thePresence of Zinc Bacitracin

The inventors then tested the effect of zinc bacitracin on the in vitrogrowth kinetics of L. major promastigotes. For this study, only zincbacitracin was tested, firstly because it had the same rLmPDI enzymaticactivity inhibition profile as bacitracin, and secondly becausebacitracin is more stable and less toxic when coupled with zinc.

To this end, promastigotes from the GLC94 isolate were cultured on amedium based on coagulated rabbit serum for two days. Then the parasites(2×10⁶ parasites per ml) were transferred into complete mediumcomprising zinc bacitracin BACZn, in concentrations of 1, 1.5 and 2.5mM. Promastigotes cultured in complete medium in the absence ofinhibitors were used as the control. Monitoring was by counting theparasites at 48, 72 and 96 hours. The results are shown in FIG. 13.

These results show that zinc bacitracin partially inhibits the growth ofparasites at 1.5 mM with complete inhibition at 2 mM and at 5 mM, whileit had no effect at 1 mM. Thus, it is very important to note that thismolecule is capable of blocking the proliferation of L. major parasitesin culture.

Example 9 Inhibition of the Growth of L. major Promastigotes in BALB/cMice in the Presence of Zinc Bacitracin

The availability of zinc bacitracin, which already forms a weapon in thetherapeutic arsenal, has allowed it to be tested on the evolution ofinfection in the BALB/c mouse with L. major. Mice were infected withpromastigotes in the stationary growth phase (10⁶ promastigotes per paw)of the GLC94 isolate into the plantar pad of the rear paw and treatedwith a pomade based on 5 mM or 25 mM of BACZn (prepared in Vaseline).Treatment with the pomade was started 48 hours after injecting theparasites, by dint of one application per day over 5 days of the week.Mice infected in the same manner and treated with Vaseline were used asthe control. The lesion size was measured each week. The results areshown in FIG. 14.

Although preliminary, these results show that zinc bacitracin attenuatesthe progress of the disease when it is applied locally in the form of apomade, at the injection site, to BALB/c mice. It should be emphasizedthat in the group of treated mice, lesion attenuation was observed in 2out of 3 mice treated with 5 mM bacitracin and 2 out of 4 mice treatedwith 25 mM bacitracin. Recurrence of the clinical disease after stoppingthe treatment was expected since BALB/c mice are incapable of completelyeliminating the parasite and even the treatments used in man (glucantimeand paramomycin) have little effect on the disease induced in the BALB/cmouse, in which complete disappearance of the parasites has never beenobserved.

LmPDI can thus be considered to be a potential target foranti-leishmania chemotherapy and it appears that bacitracin ispotentially effective against L. major.

REFERENCES

-   Beverley, S. M. and S. J. Turco (1998). “Lipophosphoglycan (LPG) and    the identification of virulence genes in the protozoan parasite    Leishmania.” Trends Microbiol(1): 35-40.-   Chakrabarty, R., S. Mukherjee, et al. (1996). “Kinetics of entry of    virulent and avirulent strains of Leishmania donovani into    macrophages: a possible role of virulence molecules (gp63 and LPG).”    J Parasitol 82(4): 632-5.-   Cotrim, P. C., L. K. Garrity, et al. (1999). “Isolation of genes    mediating resistance to inhibitors of nucleoside and ergosterol    metabolism in Leishmania by overexpression/selection.” J Biol Chem    274(53): 37723-30.-   De, T. and S. Roy (1999). “Infectivity and attenuation of Leishmania    donovani promastigotes: association of galactosyl transferase with    loss of parasite virulence.” J Parasitol 85(1):. 54-9.-   Descoteaux, A., Y. Luo, et al. (1995). “A specialized pathway    affecting virulence glycoconjugates of Leishmania Science 269(5232):    1869-72.-   Desjardins, M. and A. Descoteaux (1997). “Inhibition of    phagolysosomal biogenesis by the Leishmania lipophosphoglycan.” J    Exp Med 185(12): 2061-8.-   Desjardins, M. and A. Descoteaux (1998). “Survival strategies of    Leishmania donovani in mammalian host macrophages. Res Immunol    149(7-8): 689-92.-   Dumas, C., M. Ouellette, et al. (1997). “Disruption of the    trypanothione reductase gene of Leishmania decreases its ability to    survive oxidative stress in macrophages.” Embo J 16(10): 2590-8.-   Ferrari, D. M. and H. D. Soling (1999). “The protein    disulphide-isomerase family: unravelling a string of folds.” Biochem    J 339(Pt 1): 1-10.-   Frand, A. R., J. W. Cuozzo, et al. (2000). “Pathways for protein    disulphide bond formation.” Trends Cell Biol 10(5): 203-10.-   Garami, A. and T. IIg (2001). “The role of phosphomannose isomerase    in Leishmania mexicana glycoconjugate synthesis and virulence.” J.    Biol Chem 276(9): 6566-75.-   Gilbert, H. F. (1998). “Protein disulfide isomerase.” Methods    Enzymol 290: 26-50.-   Heard, P. L., C. S. Lewis, et al. (1996). “Leishmania mexicana    amazonensis: differential display analysis and cloning of mRNAs from    attenuated and infective forms.” J Eukarvot Microbiol 43(5): 409-15.-   Hubel, A., S. Krobitsch, et al. (1997). “Leishmania major Hsp100 is    required chiefly in the mammalian stage of the parasite.” Mol Cell    Biol 17(10): 5987-95.-   Hultgren, S. J., S. Abraham, et al. (1993). “Pilus and nonpilus    bacterial adhesins: assembly and function in cell recognition.” Cell    73(5): 887-901.-   Ilg, T. (2000). “Proteophosphoglycans of Leishmania.” Parasitol    Today 16(11): 489-97.-   Ilg, T., M. Demar, et al. (2001). “Phosphoglycan Repeat-deficient    Leishmania mexicana Parasites Remain Infectious to Macrophages and    Mice.” J Biol Chem 276(7): 4988-97.-   Kebaïer, C., H. Louzir, et al. (2001). “Heterogeneity of wild    Leishmania major isolates in experimental murine pathogenicity and    specific immune response.” Infection and Immunity 69(8).-   Khalil, E. A., A. M. E I Hassan, et al. (2000). “Autoclaved    Leishmania major vaccine for prevention of visceral leishmaniasis: a    randomised, double-blind, BCG-controlled trial in Sudan.” Lancet    356(9241): 1565-9.-   Liang, P., D. Bauer, et al. (1995). “Analysis of altered gene    expression by differential display.” Methods Enzymol 254:_(—)304-21.-   Liang, P. and A. B. Pardee (1992). “Differential display of    eukaryotic messenger RNA by means of the polymerase chain reaction.”    Science 257(5072): 967-71.-   Lira, R., S. Sundar, et al. (1999). “Evidence that the high    incidence of treatment failures in Indian kala-azar is due to the    emergence of antimony-resistant strains of Leishmania donovani.” J    Infect Dis 180(2): 564-7.-   Louzir, H., P. C. Melby, et al. (1998). “Immunologic determinants of    disease evolution in localized cutaneous leishmaniasis due to    Leishmania major.” J Infect Dis 177(6): 1687-95.-   Lyles, M. M. and H. F. Gilbert (1991). “Catalysis of the oxidative    folding of ribonuclease A by protein disulfide isomerase: dependence    of the rate on the composition of the redox buffer.” Biochemistry    30(3): 613-9.-   Lyles, M. M. and H. F. Gilbert (1991). “Catalysis of the oxidative    folding of ribonuclease A by protein disulfide isomerase:    pre-steady-state kinetics and the utilization of the oxidizing    equivalents of the isomerase.” Biochemistry 30(3): 619-25.-   Martin, J. L. (1995). “Thioredoxin—a fold for all reasons.”    Structure 3(3): 245-50.-   McKerrow, J. H., J. C. Engel, et al. (1999). “Cysteine protease    inhibitors as chemotherapy for parasitic infections.” Bioorg Med    Chem 7(4): 639-44.-   Mottram, J. C., D. R. Brooks, et al. (1998). “Roles of cysteine    proteinases of trypanosomes and Leishmania in host-parasite    interactions.” Curr. Opin Microbiol 1(4): 455-60.-   Mottram, J. C., A. E. Souza, et al. (1996). “Evidence from    disruption of the Imcpb gene array of Leishmania mexicana that    cysteine proteinases are virulence factors.” Proc Natl Acad Sci USA    93(12): 6008-13.-   Mou, Y., H. Ni, et al. (1998). “The selective inhibition of beta 1    and beta 7 integrin-mediated lymphocyte adhesion by bacitracin.” J    Immunol 161(11): 6323-9.-   Mukhopadhyay, S., P. Sen, et al. (1998). “Reduced expression of    lipophosphoglycan (LPG) and kinetoplastid membrane protein (KMP)-11    in Leishmania donovani promastigotes in axenic culture.” J.    Parasitol 84(3): 644-7.-   Noiva, R. (1999). “Protein disulfide isomerase: the multifunctional    redox chaperone of the endoplasmic reticulum.” Semin Cell Dev Biol    10(5): 481-93.-   Orlandi, P. A. (1997). “Protein-disulfide isomerase-mediated    reduction of the A subunit of cholera toxin in a human intestinal    cell line.” J. Biol. Chem 272(7): 4591-9.-   Osterneier, M., K. De Sutter, et al. (1996). “Eukaryotic protein    disulfide isomerase complements Escherichia coli dsbA mutants and    increases the yield of a heterologous secreted protein with    disulfide bonds.” J Biol Chem 271(18): 10616-22.-   Paramchuk, W. J., S. O. Ismail, et al. (1997). “Cloning,    characterization and overexpression of two iron superoxide dismutase    cDNAs from Leishmania chagasi: role in pathogenesis.” Mol Biochem    Parasitol 90(1): 203-21.-   Peek, J. A. and R. K. Taylor (1992). “Characterization of a    periplasmic thiol:disulfide interchange protein required for the    functional maturation of secreted virulence factors of Vibrio    cholerae.” Proc Natl Acad Sci USA 89(13): 62104.-   Perez-Victoria, J. M., F. J. Perez-Victoria, et al. (2001).    “High-affinity binding of silybin derivatives to the    nucleotide-binding domain of a Leishmania tropica    P-glycoprotein-like transporter and chemosensitization of a    multidrug-resistant parasite to daunomycin.” Antimicrob Agents    Chemother 45(2): 439-46.-   Ryser, H. J., E. M. Levy, et al. (1994). “Inhibition of human    inmunodeficiency virus infection by agents that interfere with    thiol-disulfide interchange upon virus-receptor interaction.” Proc    Natl Acad Sci USA 91(10): 4559-63.-   Ryan, K. A., L. A. Garraway, et al. (1993). “Isolation of virulence    genes directing surface glycosyl-phosphatidylinositol synthesis by    functional complementation of Leishmania.” Proc Natl Acad Sci USA    90(18): 8609-13.-   Sacks, D. I., G. Modi, et al. (2000). “The role of phosphoglycars in    Leislimania-sand fly interactions.” Proc Natl Acad Sci USA 97(1):    406-11.-   Selzer, P. M., X. Chen, et al. (1997). “Leishmania major: molecular    modeling of cysteine proteases and prediction of new non peptide    inhibitors.” Exp ParasitoI87(3): 212-21.-   Sharifi, I., A. R. FeKri, et al. (1998). “Randomised vaccine trial    of single dose of killed Leishmania major plus BCG against    anthroponotic cutaneous leishmaniasis in Bam, Iran.” Lancet    351(9115): 1540-3.-   Spath, G. F., L. Epstein, et al. (2000). “Lipophosphoglycan is a    virulence factor distinct from related glycoconjugates in the    protozoan parasite Leishmania major.” Proc Natl Acad Sci USA 97(16):    9258-63.-   Streit, J. A., T. J. Recker, et al. (2001). “Protective immunity    against the protozoan Leishmania chagasi is induced by subclinical    cutaneous infection with virulent but not avirulent organisms.” J    Immunol 66(3): 1921-9.-   Titus, R. G., F. J. Gueiros-Filho, et al. (1995). “Development of a    safe live leishmania vaccine line by gene replacement.” Proc Natl    Acad Sci USA 92(22): 10267-71.-   Wang, Y., E. S. Bjes, et al. (2000). “Molecular aspects of    complement-mediated bacterial killing. Periplasmic conversion of C9    from a protoxin to a toxin.” J Biol Chem 275(7): 4687-92.-   Wiese, M. (1998). “A mitogen-activated protein (MAP) kinase    homologue of Leishmania mexicana is essential for parasite survival    in the infected host.” Embo J 17(9): 2619-28.-   Yu, J. (1998). “Inactivation of DsbA, but not DsbC and DsbD, affects    the intracellular survival and virulence of Shigella flexneri.”    Infect Immun 66(8): 3909-17.-   Yu, J., B. Edwards-Jones, et al. (2000). “Key role for DsbA in    cell-to-cell spread of Shigella flexneri, permitting secretion of    Ipa proteins into interepithelial protrusions.” Infect Immun 68(11):    6449-56.-   Yu, J. and J. S. Kroll (1999). “DsbA: a protein-folding catalyst    contributing to bacterial virulence.” Microbes Infect 1(14): 1221-8.-   Yu, J., H. Webb, et al. (1992). “A homologue of the Escherichia coli    DsbA protein involved in disulphide bond formation is required for    enterotoxin biogenesis in Vibrio cholerae.” Mol Microbiol 6(14):    1949-58.-   Zhang, H. Z. and M. S. Donnenberg (1996). “DsbA is required for    stability of the type IV pilin of enteropathogenic escherichia    coli.” Mol Microbiol 21(4): 787-97.-   Zhang, W. W. and G. Matlashewski (1997). “Loss of virulence in    Leishmania donovani deficient in an amastigote-specific protein,    A2.” Proc Natl Acad Sci USA 94(16): 8807-11.

1. A protein involved in the virulence of Leishmania, comprising atleast one site (Cys-Gly-His-Cys) identical to the potential active siteof a protein from the protein disulfide-isomerase family (PDI).
 2. ALeishmania protein involved in the virulence of the parasite, comprisingat least one site (Cys-Gly-His-Cys) identical to the potential activesite of a protein from the protein disulfide-isomerase family (PDI). 3.A protein according to claim 1 or claim 2, characterized in that it isthe LmPDI protein of Leishmania major, with sequence SEQ ID No: 2, orany functional variant of LmPDI having at least 40% identity, preferablyat least 80% identity with LmPDI.
 4. A recombinant polypeptidecomprising at least one fragment of more than 10 amino acids of aprotein according to any one of claims 1 to 3, said recombinantpolypeptide being capable of triggering an immunological reactionagainst an epitope of LmPDI when administered to a human or animal host.5. A recombinant polypeptide according to claim 4, characterized in thatit is the LmPDI-(His)₆ protein with sequence SEQ ID No:
 3. 6. A fusionprotein comprising a recombinant polypeptide according to claim 4, fusedwith a further polypeptide fragment, said fusion protein being capableof triggering an immunological reaction against an LmPDI epitope when itis administered to a human or animal host.
 7. A recombinant nucleic acidsequence coding for a protein or a polypeptide according to any one ofclaims 1 to
 6. 8. A nucleic acid sequence according to claim 7,characterized in that it comprises the coding sequence corresponding tonucleotides 241 to 1674 of sequence SEQ ID No: 1, or a fragment of saidsequence 100 nucleotides or more in size.
 9. A nucleic acid vector,characterized in that it comprises a nucleic acid sequence according toclaim 7 or claim
 8. 10. A vector according to claim 9, characterized inthat it is a plasmid, a cosmid, a phage or a virus.
 11. A cultured cellcomprising a vector according to claim 9 or claim
 10. 12. A cellaccording to claim 11, characterized in that it is bacterial strainLmPDI-XL₁ deposited at the Collection Nationale de Culture desMicroorganismes [CNCM, the National Collection of MicroorganismCultures], on 31/01/2002 with accession number I-2621.
 13. Use of anucleic acid probe specifically hybridizing under highly stringentconditions with the nucleic acid sequence of SEQ ID No: 2, to determinethe presence or absence of the virulence gene coding for LmPDI in abiological sample.
 14. A nucleotide primer, characterized in that itallows specific amplification of at least a portion of the sequence ofSEQ ID No: 1, from cells infected with Leishmania, thus allowing thepresence or absence of the virulence gene coding LmPDI to be determinedin a biological sample.
 15. A purified antibody, specificallyrecognizing LmPDI.
 16. An immunogenic composition comprising a proteinaccording to claim 1, 2, 3 or 6 and/or a recombinant polypeptideaccording to claim 4 or claim 5, and/or a nucleic acid sequenceaccording to claim 7 or claim 8, and/or a vector according to claim 9 orclaim 10, and/or a cell according to claim 11, said immunogeniccomposition being capable of in vitro stimulation of the proliferationof mononuclear cells originating from individuals who have come intocontact with a Leishmania parasite.
 17. An immunogenic compositionaccording to claim 16, capable of in vitro stimulation of theproliferation of mononuclear cells originating from individuals who havecome into contact with Leishmania major.
 18. An immunogenic compositionaccording to claim 16 or claim 17, having a pharmaceutically acceptableformulation for administration to a human or animal host.
 19. Animmunogenic composition according to claims 16 to 18, capable ofinducing an immune response of the Th1 type when administered to a humanor animal host.
 20. A vaccinating composition comprising a proteinaccording to claim 1, 2, 3 or 6 and/or a recombinant polypeptideaccording to claim 4 or claim 5, and/or a nucleic acid sequenceaccording to claim 7 or claim 8, and/or a vector according to claim 9 orclaim 10, and/or a cell according to claim 11, said vaccinatingcomposition being intended to protect a human or animal host againstleishmaniasis.
 21. A vaccinating composition according to claim 20,having a pharmaceutically acceptable formulation for administration to ahuman or animal host.
 22. An immunogenic and/or vaccinating compositionaccording to any one of claims 16 to 21, further comprising an antigenforeign to Leishmania and/or a nucleic acid sequence coding for anantigen foreign to Leishmania.
 23. A method for screening molecules thatare susceptible of inhibiting the growth of Leishmania major, comprisinga step for evaluating the capacity of said molecules to inhibit theactivity of LmPDI.
 24. A screening method according to claim 23, inwhich the step for evaluating the capacity of a molecule to inhibit theactivity of LmPDI is carried out in a test for reactivating scrambledRNase A comprising the following steps: incubating scrambled RNase A inthe presence of LmPDI under conditions allowing its reactivation;incubating scrambled RNase A under conditions identical to thoseallowing its reactivation by LmPDI, the molecule to be tested beingadded; comparing the results obtained in the absence and in the presenceof the test molecule, a fault in the reactivation of RNase A in thepresence of the test molecule revealing that said molecule has an LmPDIinhibiting activity.
 25. A screening method according to claim 23 orclaim 24, further comprising a test for inhibiting the growth ofLeishmania major in a liquid medium and if appropriate a test forinhibiting the growth of Leishmania major in an experimental murinemodel of leishmaniasis.
 26. Use of one or more proteindisulfide-isomerase (PDI) inhibitors, for the preparation of apharmaceutical composition intended for prophylaxis, attenuation ortreatment of infection with Leishmania.
 27. Use according to claim 26,in which a PDI inhibitor is an anti-PDI or anti-LmPDI antibody,bacitracin, zinc bacitracin, 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB),p-chloromercuribenzene sulfonic acid (pCM3S) or tocinoic acid.
 28. Useaccording to claim 26 or claim 27, for the preparation of a compositionfor topical, oral or parenteral administration to a human or animalhost.
 29. Use of bacitracin or zinc bacitracin as an inhibitor of thegrowth of a parasite responsible for leishmaniasis or as an active agentagainst a leishmaniasis infection.
 30. A pharmaceutical compositionintended for the treatment of a leishmaniasis infection, comprising anantibody according to claim
 15. 31. A composition according to claim 30,suitable for topical, oral or parenteral administration.
 32. Apharmaceutical composition intended for the treatment of an infectionwith Leishmania, containing one or more protein disulfide-isomerase(PDI) inhibitors.
 33. A pharmaceutical composition according to claim32, containing bacitracin or zinc bacitracin.
 34. An in vitro method fordiagnosing an infection by a parasite responsible for leishmaniasis,characterized in that it comprises: bringing at least one antibodyaccording to claim 15 into contact with a biological sample from asubject partially infected with a parasite responsible for leishmaniasisunder conditions allowing the formation of an immune complex betweensaid antibody and antigenic proteins contained in the sample; detectingsaid complex.
 35. A diagnostic kit for carrying out the method accordingto claim 34, characterized in that it comprises: at least one antibodyaccording to claim 15; a medium suitable for forming an immune complexwith said antibody; reagents allowing the detection of any complexesthat are formed; control samples, if appropriate.